During these studies morphine was observed to have unexpected effects on the endocytic membrane trafficking of the mu opioid receptor (MOR) in physiologically relevant medium spiny neurons in the rat Nucleus Accumbens and in primary culture, and unexpected regulatory effects on endosome recruitment of non-visual (beta-) arrestins were also observed in dendrites of these neurons. The proposed studies seek to elucidate the mechanistic basis of MOR regulatory effects in medium spiny neurons, and to test specific hypotheses regarding possible regulatory functions on opioid and co-expressed dopamine receptors relevant to mesolimbic dopamine function and the rewarding effects of opioid drugs. The Specific Aims of the proposed studies are to: (1) Define mechanisms of morphine-induced endocytosis of MOR and endosome recruitment of beta- arrestins in medium spiny neurons. The proposed studies seek to elucidate the mechanistic basis of these unprecedented morphine effects. The hypothesis to be tested is that MOR endocytosis and endosome recruitment of beta-arrestin is a consequence of specific cytoplasmic phosphorylation(s) in MOR. (2) Identify mechanisms of D1 dopamine receptor regulation in relevant MOR-expressing neurons. MOR- expressing medium spinal neurons are major targets of VTA dopaminergic signaling via co-expressed D1 receptors (D1R). Preliminary studies suggest that drug-mediated activation of MOR, by essentially 'sequestering'beta-arrestins on endosomes, may attenuate arrestin-dependent D1R endocytosis induced by dopamine. This hypothesis will be tested using site-directed mutagenesis and established co-transfection methods in rat striatal neurons. (3) Utilize live cell imaging methods to visualize drug effects on relevant receptor and arrestin trafficking events in real time. GFP tagging and live cell imaging methods to the regulated trafficking of MOR in rat striatal neurons, focusing on spatial aspects of MOR recycling following drug-mediated activation.